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首页> 外文学位 >The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases.
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The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases.

机译：氨基酰基-tRNA合成酶在编辑和复合酶复合物形成的翻译中的作用。

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摘要

Aminoacyl-tRNA synthetases (aaRSs) catalyze the first step of translation, aminoacylation. These enzymes attach amino acids (aa) to their cognate tRNAs to form aminoacyl-tRNA (aa-tRNA), an important substrate in protein synthesis, which is delivered to the ribosome as a ternary complex with translation elongation factor 1A (EF1A) and GTP. All aaRSs have an aminoacylation domain, which is the active site that recognizes the specific amino acid, ATP, and the 3 ' end of the bound tRNA to catalyze the aminoacylation reaction. Apart from the aminoacylation domain, some aaRSs have evolved additional domains that are involved in interacting with other proteins, recognizing and binding the tRNA anticodon, and editing misacylated tRNA thereby expanding their role in and beyond translation.;One such function of the aaRS is to form a variety of complexes with each other and with other factors by interacting via additional N or C terminal extensions. For example, several archaeal and eukaryotic aaRSs are known to associate with EF1A or other aaRSs forming higher order complexes, although the role of these multi-synthetase complexes (MSC) in translation remains largely unknown. MSC function was hence investigated in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aaRS activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These in vivo data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies and could potentially increase translation efficiency by limiting substrate diffusion from the ribosome, thus facilitating rapid recycling of tRNAs.;In addition to their aminoacylation functions, about half of the aaRSs have evolved an editing function, which hydrolyzes non-cognate amino acid from its cognate tRNA thereby maintaining the fidelity of translation. Phenylalanyl-tRNA synthetase (PheRS) misactivates and mischarges Tyr onto tRNAPhe, but is able to correct the mistake using a proofreading activity which hydrolyzes mischarged Tyr-tRNAPhe. The requirement for PheRS editing and fidelity of Phe codon translation is specific for different cellular compartments in eukaryotes and varies significantly within bacteria. Yeast cytoplasmic PheRS (ctPheRS) has a low Phe/Tyr specificity and is capable of editing, whereas the yeast mitochondrial enzyme (mtPheRS) completely lacks an editing domain, and instead relies on high Phe/Tyr specificity. Escherichia coli, in contrast, has retained features of both yeast enzymes and displays a high degree of Phe/Tyr specificity and robust editing activity. We showed that in E. coli the editing domain has evolved to efficiently edit m-Tyr-tRNA Phe, and that this editing activity is essential for cellular growth and viability in the presence of the non-proteinogenic amino acid m- Tyr and oxidative stress conditions. In comparison, in the yeast enzyme, due to the low specificity of its active site, cytoplasmic PheRS editing has evolved to protect the proteome from p-Tyr misincorporation as shown by the requirement for editing activity to survive in the presence of high concentrations of Tyr compared to Phe. Hence different environmental factors and cell physiology drive the selection of quality control mechanisms in various organisms.;While PheRS has evolved to possess editing activity to actively edit noncognate amino acids (both proteinogenic and non-proteinogenic), editing mechanisms are not evolutionarily conserved. Tyrosyl-tRNA synthetases are among the aaRSs lacking any known editing activity. The high specificity displayed by this aaRSs is achieved by taking advantage of the unique structural and chemical properties of certain amino acids, leading to favorable binding affinities of cognate over non-cognate substrates in the active site of the enzyme. Its cognate amino acid Tyr differs from Phe by a single hydroxyl group, and the specific recognition and binding of the hydroxyl group allows bacterial TyrRS to discriminate against non-cognate Phe with a specificity of 105. However, recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. We examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with the decrease of Tyr in the amino acid pools, suggesting that mischarging of tRNATyr with non-cognate Phe by TyrRS was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic TyrRS revealed a twenty five-fold lower specificity for Tyr over Phe compared to previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial TyrRSs revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.
机译：氨酰基-tRNA合成酶（aaRSs）催化翻译的第一步，氨基酰化。这些酶将氨基酸（aa）附着到它们的同源tRNA上以形成氨酰基tRNA（aa-tRNA），这是蛋白质合成中的重要底物，以具有翻译延伸因子1A（EF1A）和GTP的三元复合物的形式传递到核糖体中。 。所有aaRS均具有氨基酰化结构域，其是识别特定氨基酸，ATP和结合的tRNA的3'端以催化氨基酰化反应的活性位点。除了氨基酰化结构域外，一些aaRS还进化了其他结构域，该结构域与其他蛋白质相互作用，识别并结合tRNA反密码子并编辑错误的酰化tRNA，从而扩展了它们在翻译中和翻译后的作用。通过其他N或C末端延伸基团相互作用，彼此之间以及与其他因素形成多种络合物。例如，尽管这些多合成酶复合物（MSC）在翻译中的作用仍是未知之数，但已知几种古细菌和真核aaRS与EF1A或其他形成较高阶复合物的aaRS相关。因此，在古生嗜热球菌中研究了MSC的功能，其中六个aaRS与蛋白质合成中涉及的其他几种因子亲和共纯化，表明MSC可能与翻译的核糖体直接相互作用。为支持该假设，MSC的aaRS活性在分离的T. kodakarensis多核糖体级分中富集。这些体内数据表明，古细菌蛋白质合成机制的成分结合到大分子装配中，并可能通过限制底物从核糖体的扩散来潜在地提高翻译效率，从而促进tRNA的快速回收。 aaRSs已进化出一种编辑功能，该功能可从其同源tRNA水解非同源氨基酸，从而保持翻译的保真度。苯丙氨酰-tRNA合成酶（PheRS）会使Tyr失活并错位到tRNAPhe上，但能够利用校对活性来纠正错误，该校对活性可以水解错位的Tyr-tRNAPhe。对PheRS编辑和Phe密码子翻译保真度的要求特定于真核生物中的不同细胞区室，并且在细菌内差异很大。酵母胞质PheRS（ctPheRS）具有较低的Phe / Tyr特异性，并具有编辑能力，而酵母线粒体酶（mtPheRS）完全缺乏编辑域，而依赖于高Phe / Tyr特异性。相比之下，大肠杆菌保留了两种酵母酶的功能，并显示出很高的Phe / Tyr特异性和强大的编辑活性。我们表明，在大肠杆菌中，编辑域已经进化为可以有效编辑m-Tyr-tRNA Phe，并且这种编辑活性对于存在非蛋白质氨基酸m-Tyr和氧化应激的细胞生长和活力至关重要。条件。相比之下，在酵母酶中，由于其活性位点的特异性较低，已发展出细胞质PheRS编辑以保护蛋白质组免受p-Tyr错掺，这表明需要编辑活性才能在高浓度Tyr存在下生存比起Phe。因此，不同的环境因素和细胞生理学驱动着各种生物中质量控制机制的选择。虽然PheRS已发展为具有编辑活性以主动编辑非同源氨基酸（蛋白原性和非蛋白原性氨基酸），但进化机理却不保守。酪氨酰-tRNA合成酶是缺乏任何已知编辑活性的aaRS之一。通过利用某些氨基酸的独特结构和化学特性，可以实现此aaRSs的高特异性，从而在酶的活性位点上导致同源物相对于非同源底物具有有利的结合亲和力。它的同源氨基酸Tyr与Phe的区别在于单个羟基，并且羟基的特异性识别和结合使细菌TyrRS可以区分非同源Phe，特异性为105。但是，最近的研究表明错误率可能实际上，在不同增长条件下的翻译过程中，差异很大。我们检查了酪氨酸限制的中国仓鼠卵巢（CHO）细胞中产生的重组抗体的合成过程中，Tyr密码子上Phe的错误掺入。发现Tyr到Phe的替换在整个抗体中的发生率高达0.7％，而与翻译的Tyr密码子的身份或背景无关。对Phe和Tyr水平的监测表明，错误率的变化与氨基酸库中Tyr的减少有关，表明TyrRS将tRNATyr与非同源Phe误充电是造成翻译错误的原因。 CHO细胞质TyrRS的稳态动力学分析表明，与以前鉴定的细菌酶相比，与Phe相比，Tyr的特异性降低了25倍，这与在酪氨酸限制酶过程中观察到的翻译错误率增加是一致的。哺乳动物和细菌TyrRS的功能比较揭示了负责氨基酸识别的残基的关键差异，突显了翻译质量控制的进化约束条件上的差异。

著录项

  • 作者

    Raina, Medha Vijay.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Biochemistry.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 207 p.
  • 总页数 207
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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